Sindbis virus is a member of the Alphavirus family which contains a number of human and animal pathogens. The goal of the proposal is to obtain an understanding at the molecular level of the events involved in the initiation of Sindbis virus minus strand RNA synthesis. An in vitro replication system will be used to examine the minimum cis-acting requirements for Sindbis virus minus-strand RNA synthesis. Using small template RNAs representing the minimal template for minus strand RNA synthesis mutagenesis, template competition and crosslinking assays will be used to determine the requirements for template recognition. Biochemical analyses using inhibitors of RNA synthesis will be aimed at determining the stability of the initiating and elongating replicase complexes. Finally using various template RNAs the role of sequences other than the minimal promoter in minus strand synthesis will be determined. These studies will be extended to plus-strand RNA synthesis.